The biochemical mechanism for methionine dependency has been studied extensively, but its effect on tumors derived from the sympathetic nervous system remains unclear. At high concentrations of rMETase, our studies showed that all NB cell lines will eventually die. At intermediate concentrations some will survive and some will linger on. Cells can be salvaged (death prevented) when methionine is added back to the medium, suggesting that methionine depletion is the major mechanism of cell death. We interpret these findings to mean that there is differential sensitivity of NB cell lines to methionine deprivation, as well as differences in their inherent resistance to apoptosis and cell death. We have looked for apoptosis at 24, 48 and 72 hours of rMETase treatment, and not beyond because massive cell loss usually has occurred by day 2. Observation at 48 hours after treatment with rMETase seemed to be most optimal time point for showing apoptosis. But we could document apoptosis in only 1 of 15 cell lines. We concluded that apoptosis is not the primary mechanism of cell death in the majority of NB cell lines tested here. This defect in apoptosis may relate to their caspase 3 and 8 deficiency,34
especially when MYCN
was amplified, a genetic aberration typically found in these cell lines.
Methionine deprivation modulates the expression levels of many genes in tumor cells, 16, 17
some of which can provide rationales for combination approaches utilizing rMETase and chemotherapeutic drugs. We therefore undertook an analysis of standard chemotherapeutic agents against NB where the mechanisms of drug action were known. We tested for the presence of synergy between these chemotherapeutic agents and methionine deprivation using rMETase. We observed strong synergism between rMETase and vincristine, an M phase specific agent. We expanded these synergy studies using a panel of M phase specific agents (vincristine, vinorelbine, vinblastine, mebendazole, paclitaxel, docetaxel, and iso-fludelone) and found synergy only for microtubule depolymerization agents and not microtubule stabilizing agents. The mechanism of action likely involves both the methylation of tubulins and regulation of mitosis related gene by rMETase. S-adenosyl-L-methionine (SAM), produced from methionine and ATP, is the universal methyl donor in prokaryotes and eukaryotes and reduction of SAM compromises protein methylation. Methionine appears to be critical for the methylation of actins and tubulins in maintaining cellular stability35
and SAM is known to stabilize microtubule polymerization and reduce micronuclei formation.36
Thus, it is likely rMETase reduces methylation of tubulins, further destabilizing microtubules when tumor cells are subjected to depolymerization agents. On the other hand, rMETase can up-regulate (CDKN1A, and MDA1, etc) or down-regulate(CDKN2B, and Aurora Kinase B, etc).16, 17
The change in their genes expression levels is associated with the loss of mitosis in tumor cells.16
rMETase-induced genetic alteration shows the multifaceted effect of methionine deprivation,16, 17
which can explain the highly effective synergism between rMETase and a variety of diverse chemotherapies tested.
Downregulation of O6-methylguanine-DNA methyltransferase37
and reduction of SAM by methionine deprivation will decrease DNA methylation and affect DNA stability. Less stability of DNA increases the sensitivity of tumor cells to DNA damaging agents.38
When chemotherapeutic drugs were combined with methionine depletion, cell lines established at diagnosis had substantially smaller CIs and higher sensitivity than those established at relapse. It is highly likely that these resistant cell lines have already recruited pathways to repair DNA damage.39
One might expect the combination of rMETase and DNA damaging agents to be most useful if DNA repair pathways are inhibited. Poly(ADP-ribose) polymerase (PARP) inhibitor is cytotoxic for homologous-recombination (HR)-deficient cells by inhibiting base-excision repair.40
Our preliminary results suggested that PARP inhibitor synergized with rMETase in suppressing the proliferation of NB cell lines. A more detailed synergism analysis using cell lines established at diagnosis versus at relapse, with known DNA damage repair pathways will be critical for understanding the underlying mechanism of drug resistance.
For patients with high risk NB, acquired resistance to chemotherapy and the toxicity of chemotherapy in young children are clinical constraints. Late effects such as second cancer are clearly related to dose and intensity of chemoradiotherapy. Overcoming drug resistance or lowering drug doses to minimize toxicity can have a significant impact on patient survival. Given the toxicity profile and our results on preclinical efficacy of rMETase in human NB, methionine depletion especially when induced by rMETase may provide a useful adjunct to our current therapy of advanced stage disease. rMETase may be most effective when applied at initial diagnosis with diverse classes of chemotherapy. At relapse following exposure to chemotherapy, the synergy appeared to be restricted to microtubule depolymerization agents, a unique class of agents with primarily neurotoxicity, but minimal myelosuppression. The underlying mechanism of this cooperative effect is not currently understood. In contrast, neither microtubule stabilizing agents nor gemcitabine showed synergy despite their in vitro activity as single agents and their clinical efficacy in the treatment of NB.
In conclusion, methionine depletion inhibited NB proliferation and arrested tumor cells at G2 phase. However, apoptosis was not the primary mechanism of cell death in the majority of cell lines examined. As a single agent in vivo, methionine depletion inhibited NB xenografts growth. In combination therapy, methionine depletion synergized with microtubule depolymerization agents in vitro and rendered vincristine more effective than vincristine alone in tumor growth suppression in vivo. Synergism between methioninase and DNA damaging agents was dependent on whether cell lines were established at diagnosis or at relapse.